Research reported in this publication was supported by The Johns Hopkins Applied Physics Laboratory&#8217;s Research and Exploratory Development Department, Office of Technology Transfer, and Stuart S. Janney Fellowship Program, in addition to the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R21HL127355.

1. Processing of SLNs
<ol>
	<li>Synthesis of SLNs 
	Note: Use the PIT method 1,17,20 to prepare the sub-20 nm SLNs 1.
	<ol>
		<li>Use aseptic technique, bioreagents or cell culture grade reagents, and sterile materials (i.e., pipettes, spatulas, vials, etc.).</li>
		<li>Use a biosafety cabinet for the synthesis of SLNs (Figure 2).</li>
		<li>Combine 0.6 mg of fluorescent dye (Nile Red (NiR), NiR concentration is approximately 0.3 mg/ml) and 0.10 g of Heneicosane (Lipid, 5 wt/wt% lipid concentration) into a vial (15 ml), co-melt at 90 &#176;C, and stir.</li>
		<li>Add 0.11 g polyoxyethylene (10) oleyl ether (Surfactant, 5.5 wt/wt% surfactant concentration). Co-melt the resulting mixture at 90 &#176;C and stir.</li>
		<li>Add 1.79 g of sterile water to the mixture, heat to 90 &#176;C, and stir until a transparent nanoemulsion is formed. 
		Note: Under continued stirring and controlled cooling, SLNs are created. These conditions cause an inversion of the water-in-oil emulsion to an oil-in-water emulsion. Lipophilic molecules such as NiR partition to the lipid droplets.</li>
		<li>Prepare a nanoemulsion in parallel without adding the fluorescent dye, in order to serve as a control sample. Combine 0.10 g of Heneicosane and 0.11 g polyoxyethylene (10) oleyl ether into a vial (15 ml) co-melt at 90 &#176;C, and stir.
		<ol>
			<li>Add 1.79 g of sterile water to the mixture, heat to 90 &#176;C, and stir until a transparent nanoemulsion is formed.</li>
		</ol>
		</li>
		<li>Further sterilize each nanoemulsion using a sterile 0.2 &#181;m filter.</li>
	</ol>
	<img alt="Figure 2" src="/files/ftp_upload/53102/53102fig2.jpg" /> 
	Figure 2. Schematic of synthesis of SLNs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a></li>
	<li>Particle Size and Polydispersity
	<ol>
		<li>Use dynamic light scattering (DLS) to measure the particle size and polydispersity of the SLNs 1 using a glass cuvette and an instrument designed to measure particle size.</li>
		<li>Prior to the measurements of the samples, measure the particle sizes of two known standards following the manufacturer&#8217;s protocol for the standards preparation and measurement.</li>
		<li>Fill the glass cuvette with 1.5 ml of standard solution and measure the standard solution using the same measurement conditions as the samples. 
		Note: Previous step was repeated for each standard.</li>
		<li>Measure the samples as prepared. For each, use the following experimental parameters: a run time of 100 sec, refractive index of water (1.33), viscosity of water at 20 &#176;C (1.002 mPa&#183;sec). 
		Note: This technique uses frequency shifted light to measure the size of the nanoparticles.</li>
		<li>Report the average particle diameter and polydispersity of particle size distribution.</li>
	</ol>
	</li>
	<li>Melting Point and Latent Heat of Melting
	<ol>
		<li>Use a differential scanning calorimeter (DSC) equipped with an auto-sampler and liquid nitrogen cooling source to investigate the thermal behavior of SLNs 1.</li>
		<li>Pipette the as-prepared SLNs into a 40 &#181;l aluminum pan with a mass of approximately 25 mg and hermetically seal the pan using a universal crimper press to minimize moisture loss during the DSC scan.</li>
		<li>Measure each nanoemulsion from 5 to 80 &#176;C.</li>
		<li>Report the melting point and the latent heat of melting from the DSC plot, where the valley represents the melting point and the integral of the area under the curve in the DSC plot divided by the amount of material represents the latent heat of melting.</li>
	</ol>
	</li>
</ol>
2. SLNs Interaction with Fibroblasts and Dendritic Cells
<ol>
	<li>Culture of Primary Human Fibroblasts
	<ol>
		<li>Culture primary human fibroblasts according to the manufacturer&#8217;s instructions in liquid medium for the culture of human dermal fibroblasts (medium 106), supplemented with the low serum growth supplement (LSGS) kit. Maintain cells in a humidified atmosphere at 37 &#176;C, 5% CO2 and subculture upon reaching 80% confluence.</li>
		<li>For both MTT and imaging analysis, seed cells in a 96-well plate, at a density of 2 x 104 cells per well, and allow to equilibrate at 37 &#176;C O/N prior to SLN exposure.</li>
		<li>At time zero, dilute SLNs in complete medium as indicated (Figure 4), with respect to lipid concentration, and add 10 &#956;l per well to each replicate well in the 96-well plate.</li>
		<li>Maintain cells at 37 &#176;C, 5% CO2, during the incubation period.</li>
	</ol>
	</li>
	<li>MTT Toxicity Assay
	<ol>
		<li>After 24 hr of incubation with SLNs, determine cellular viability using the MTT assay, according to the manufacturer&#8217;s protocol.</li>
		<li>Wash cells once and add fresh medium to each well (100 &#956;l/well). Kill control wells by incubating in a 70% methanol solution for 10 min before replacing with fresh medium.</li>
		<li>Add MTT reagent (10 &#956;l per well) to each well of the microplate and incubate O/N at 37 &#176;C.</li>
		<li>After O/N incubation, solubilize intracellular formazin crystals with the provided detergent solution (100 &#956;l per well) according to manufacturer&#8217;s protocol.</li>
		<li>After 3 hr of incubation at RT, obtain absorbance values at a measurement wavelength of 570 nm using a microplate reader. 
		Note: MTT reagent used should be less than 1% (w/v) 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyl-tetrazolium bromide.</li>
	</ol>
	</li>
	<li>Fluorescence Microscopy
	<ol>
		<li>At specific time points following dosing of fibroblasts with SLN, wash the fibroblasts twice with sterile phosphate buffered saline (PBS) and fix for 10 min with cold 70% methanol.</li>
		<li>After 10 min, replace the methanol with PBS and capture images using an inverted microscope, using a band pass (BP) 690/50 nm filter set.</li>
	</ol>
	</li>
	<li>Culture of Mouse Bone Marrow Dendritic Cells
	<ol>
		<li>Induce mouse bone marrow-derived dendritic cells (BMDC) as previously described 23,24. Briefly, plate single cell suspensions of C57BL/10 bone marrow in 100 mm Petri dishes at 2 x 106/plate with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF, 300 U/ml) and recombinant murine IL-4 (B cell stimulating factor, 200 U/ml). Change media every 3 days.</li>
		<li>On day 7, add NiR-loaded SLNs to the dishes at a final concentration of 0.5 and 5.0 &#181;g/ml lipid and maintain the cells in a 37 &#176;C, 5% CO2 incubator for the desired length of exposure.</li>
		<li>Collect cells from the dishes by aspirating all the media in the plate, collecting it in a 50 ml tube, followed by thorough washing of the plate (to dislodge loosely adherent cells) with 5 ml of phosphate buffered saline (PBS) containing 4 mM ethylenediaminetetraacetic acid (EDTA). Collect the obtained cell suspension in the same 50 ml tube.</li>
	</ol>
	</li>
	<li>Assessment of SLNs Incorporation via Flow Cytometry
	<ol>
		<li>Determine the concentration of the cell suspension obtained in point 2.4.3 and distribute 3x105 cells into a 12 mm x&#160;75&#160;mm round bottom FACS tube.</li>
		<li>Wash the cells by adding 1 ml of PBS and then centrifuging the tubes for 5 min at 400 x g, at a temperature of 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend the pellet with 100 &#181;l of PBS/2 mM EDTA/0.1% bovine serum albumin (BSA) (staining buffer) containing anti-CD11c-FITC monoclonal antibody (0.25 &#956;g/ml final). Incubate for 30 min on ice in the dark.</li>
		<li>Wash the cells with 1 ml of PBS and then centrifuge the tubes for 5 min at 400 x g, at a temperature of 4 &#176;C.</li>
		<li>Discard the supernatant and resuspend each pellet with 400 &#181;l of staining buffer. The cells are now ready for acquisition of the samples with a flow cytometer (possibly equipped with blue and red lasers).</li>
		<li>Using flow cytometry analysis software, assess the level of incorporation of SLNs by DC by gating on the population of CD11c+ cells (FITC positive) and comparing the intensity of SLNs-associated fluorescence (measured in the PerCP-Cy5.5 or APC channels).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

In this study, the synthesis of SLNs and their applicability for intracellular targeting applications were explored. These biocompatible nanoparticles have shown promise as delivery vehicles for multiple applications including drug delivery, gene silencing, and vaccine technologies 25-30. Ultra-small SLNs were synthesized using a facile process, and their interactions with primary skin cells and primary immune cells were explored. SLNs were designed to include encapsulation of a fluorescent dye (NiR), which served as a model therapeutic cargo.
The PIT method was employed to synthesize SLN and the resulting particle properties (particle size, polydispersity, melting point, and latent heat of melting) were evaluated using dynamic light scattering and differential scanning calorimetry. Particle size and polydispersity analysis revealed the synthesis of ultra-small SLNs with narrow polydispersity, ideal for intracellular targeting applications. As previously reported, particle size can play an important role on cellular interactions 12. The size of nanoparticles has been shown to have a dramatic effect, influencing uptake efficiency, internalization pathway selection, intracellular localization, and cytotoxicity 12. Some of the overall trends with respect to cell-nanoparticle interactions documented in the literature include: critical size can vary with cell type and surface properties of the nanoparticles, and small nanoparticles have higher probability to be internalized by passive up-take than large ones 12. Thermal analysis of the particles in this study revealed a different level of crystallinity (i.e., latent heat of melting) between the control SLNs and those loaded with NiR dye. The crystallinity of the SLNs decreased due to the addition of the fluorescent dye, which acts as an impurity. The crystallinity of the therapeutic delivery system has been shown to be an important factor that affects the delivery and dose 31. A decrease in crystallinity of the SLN vehicle can influence parameters such as therapeutic loading capacity and drug release kinetics 31. The PIT method is a more gentle synthesis technique in comparison with other methods, with the limitation that only lipophilic drugs can be incorporated into the particles. Despite this limitation, SLNs synthesized using this method are highly biocompatible, making then suitable delivery platform for many applications in biomedical research.
The effects of SLN interaction on human dermal fibroblast viability were analyzed using an MTT assay. A dose-dependent decrease in viability was observed with increasing concentrations of SLN. During SLN synthesis, the lipid particles are stabilized with a surfactant layer. As the particle size decreases, surface area increases, as does the potential for cell surface interactions and cellular exposure to the surfactant layer, which may damage cell membranes. These experiments allowed the determination of the dosing concentration at which cellular uptake of particles could be observed while avoiding a significant decrease in cell viability due to membrane disruption. MTT analysis is a quantitative technique that can be broadly applied to understanding cellular health. In parallel, cells were examined using fluorescence microscopy in order to visualize the fluorescence of the cargo fluorescent dye, NiR. Using this technique, particle uptake was observed after only 2 hr of incubation with human fibroblasts. Fluorescence microscopy provides qualitative information related to cellular morphology and characteristics. Care must be taken to avoid excessive background staining and imaging of artifacts. In addition, as mouse models are generally the first choice for in depth cellular and molecular investigations, we analyzed the interaction between SLNs and murine cells. In particular, a great interest lies in the use of nanoparticles for the selective and controlled release of drugs that would modify the behavior of the immune system. Consequently, flow cytometry was employed to measure the uptake of particles by mouse dendritic cells. These data confirmed that phagocytic cells like dendritic cells can tolerate a similar range of SLNs concentration (with reduced viability observed at 50 &#181;g/ml lipid as with human dermal fibroblasts) and the level of incorporation directly correlates with the concentration of SLN exposure. Moreover, DC revealed a very rapid incorporation of SLNs, suggesting that this population might represent a valuable target via SLN-mediated drug delivery.
This study includes the description of a method for the synthesis of ultra-small populations of biocompatible nanoparticles, as well as several in vitro methods by which to assess their cellular interactions. In future studies, additional assays may be employed to further characterize the particle-cell interactions and ultimately guide the successful development of therapeutic nanocarriers. These could include studies of drug release kinetics, analysis of cellular tropism, measurement of pro-inflammatory cytokine levels, and analysis of cellular transcriptomic changes over time.

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. These vehicles can be loaded with drugs, proteins, and nucleic acids designed to impact cellular responses and achieve a desired effect in target tissues. Many types of nanocarriers have been explored for therapeutic and diagnostic benefit including lipids, polymers, silicon, and magnetic materials. These systems are attractive due to their potential for localized drug delivery, increased therapeutic concentration in target tissues, and reduction of systemic toxicity.
Solid lipid nanoparticles (SLNs) are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle in recent years. SLNs can be readily formulated for multiple applications including bio-sensing 1, cosmetics 2, and therapeutic delivery 3-7. Their utility stems from the fact that they are comprised entirely of resorbable, nontoxic lipids, resulting in enhanced biocompatibility. During synthesis, lipophilic drugs can be incorporated into SLN vehicles, thereby increasing drug solubility and suitability for parenteral administration. SLN vehicles also help to stabilize encapsulated therapeutics, reducing their degradation and clearance, and maximizing therapeutic action. These vehicles are particularly well suited for long acting, controlled-release preparations due to their stability at body temperature 3,4,8,9. Importantly, encapsulation of drugs in lipid nanoparticles alters the intrinsic pharmacokinetic profiles of the drug molecules. This provides a potential advantage by allowing the controlled release of drugs with a narrow therapeutic index. The release rate of SLN-incorporated therapeutics can be tuned based on the lipid degradation rate or the drug diffusion rate in the lipid matrix.
SLNs are often engineered to accumulate in specific target tissues. For example, their size (typically greater than 10 nm) potentiates retention in the circulation, where the leaky vasculature of tumor tissue facilitates deposition. In addition, the route of particle administration has been shown to alter biodistribution with the potential to target specific physiological structures such as lymph nodes 10,11. Upon deposition in target tissues, achieving appropriate cellular interactions and eventual internalization of nanoparticles is challenging due to the ability of cell membranes to selectively control the flow of ions and molecules into and out of the cell 12. To facilitate cellular uptake, it is possible to modify nanocarriers with specific ligands including peptides, small molecules, and monoclonal antibodies13,14. Several mechanisms including both passive penetration and active transport of nanoparticles across the cell membrane have been previously described 3,12,15. In general, it has been demonstrated that cell-nanoparticle interactions are influenced by the physicochemical properties of the nanoparticles including size, shape, surface charge and surface chemistry, in addition to cell-specific parameters such as cell type or cell cycle phase 12.
A previous investigation demonstrated the synthesis of sub-10 nm SLNs for topical 16 and biomarker detection applications 1 using the phase inversion temperature (PIT) method 17. This is a gentle synthesis method where 2 the composition remains constant while the temperature is gradually changed. Continuous stirring of the heated solution, as it cools to RT results in a nanoemulsion. This process results in the synthesis of SLNs with smaller particle size1 than that previously reported using various methods for the synthesis of lipid nanoparticles17-22. The resulting size scale, less than 20 nm, provides an advantage for intracellular targeting applications due to increased surface area and the potential for enhanced cellular interactions.
A schematic of SLNs, designed to deliver a fluorescent dye or therapeutic, is shown in Figure 1. The SLNs consist of a lipid interior (e.g., linear alkane) allowing the incorporation of lipophilic compounds (e.g., dyes or therapeutics) and a surfactant exterior (e.g., linear nonionic surfactant) surrounded by water. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. Primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time in order to characterize interactions with respect to toxicity and particle uptake. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels. Fluorescence microscopy and flow cytometry were two methods employed to examine particle uptake in vitro.
<img alt="Figure 1" src="/files/ftp_upload/53102/53102fig1.jpg" /> 
Figure 1. Schematic of SLN showing the major constituents. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Nile Red (NiR)</td>
			<td>Sigma</td>
			<td>19123</td>
			<td>BioReagent, suitable for fluorescence, &#8805;98.0%</td>
		</tr>
		<tr>
			<td>Heneicosane</td>
			<td>Aldrich</td>
			<td>286052</td>
			<td>98%</td>
		</tr>
		<tr>
			<td>Brij O10</td>
			<td>Sigma</td>
			<td>P6136</td>
			<td>Brij 97, C18-1E10, Polyoxyethylene (10) oleyl ether</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>Sterile-filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.2 &#181;m Supor Membrane Low Protein Binding</td>
			<td>Life Sciences</td>
			<td>PN4612</td>
			<td>Non-Pyrogenic</td>
		</tr>
		<tr>
			<td>Nanotrac Ultra&#160;</td>
			<td>Microtrac</td>
			<td>serial number U1985IS</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Differential Scanning Calorimeter</td>
			<td>Mettlet-Toledo</td>
			<td>----</td>
			<td>Instument</td>
		</tr>
		<tr>
			<td>Primary human fibroblasts&#160;</td>
			<td>Life Technologies</td>
			<td>C-004-5C</td>
			<td>Neonatal (HDFn)</td>
		</tr>
		<tr>
			<td>Medium 106</td>
			<td>Life Technologies</td>
			<td>M-106-500</td>
			<td>A sterile, liquid medium for the culture of human dermal fibroblasts.</td>
		</tr>
		<tr>
			<td>Low Serum Growth Supplement Kit (LSGS Kit)</td>
			<td>Life Technologies</td>
			<td>S-003-K</td>
			<td>All the components of complete LSGS</td>
		</tr>
		<tr>
			<td>MTT Cell Proliferation Assay Kit</td>
			<td>Trevigen</td>
			<td>4890-025-K</td>
			<td>Sensitive kit for the measurement of cell proliferation based upon the reduction of the tetrazolium salt, 3,[4,5-dimethylthiazol-2- yl]-2,5-diphenyl-tetrazolium bromide (MTT)</td>
		</tr>
		<tr>
			<td>Safire2 microplate reader</td>
			<td>Tecan</td>
			<td>----</td>
			<td>Instrument</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</td>
			<td>Sigma</td>
			<td>P5493</td>
			<td>For molecular biology</td>
		</tr>
		<tr>
			<td>Recombinant murine GM-CSF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>415</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
		<tr>
			<td>Recombinant murine IL-4&#160;</td>
			<td>R&#38;D Systems</td>
			<td>404</td>
			<td>&#62;97%, by SDS-PAGE under reducing conditions and visualized by silver stain.</td>
		</tr>
	</tbody>
</table>

solid lipid nanoparticles (slns) for intracellular targeting applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter cellular signaling and gene expression. In a previous investigation, the synthesis of ultra-small solid lipid nanoparticles (SLNs) for topical drug delivery and biomarker detection applications was demonstrated. SLNs are a well-studied example of a nanoparticle delivery system that has emerged as a promising drug delivery vehicle. In this study, SLNs were loaded with a fluorescent dye and used as a model to investigate particle-cell interactions. The phase inversion temperature (PIT) method was used for the synthesis of ultra-small populations of biocompatible nanoparticles. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylphenyltetrazolium bromide (MTT) assay was utilized in order to establish appropriate dosing levels prior to the nanoparticle-cell interaction studies. Furthermore, primary human dermal fibroblasts and mouse dendritic cells were exposed to dye-loaded SLN over time and the interactions with respect to toxicity and particle uptake were characterized using fluorescence microscopy and flow cytometry. This study demonstrated that ultra-small SLNs, as a nanoparticle delivery system, are suitable for intracellular targeting of different cell types.

The PIT method was used to synthesize the SLNs and the phase inversion temperature was determined utilizing a water bath. The samples were slowly heated and gently agitated until the solution appeared clear. The phase inversion temperature for the SLNs made using heneicosane lipid is 45 &#176;C. Table 1 summarizes the particle size, polydispersity, melting point and latent heat of melting for the SLNs. The SLNs synthesized using the processing conditions described above resulted in control SLNs and NiR-loaded SLNs with an average particle diameter of 18.59 and 16.87 nm and with a polydispersity of 5.83 and 4.47, respectively (Figure 3). The stability of the SLNs dispersion was monitored by the measurement of the particle size over 6 days at two different storage temperatures (4 and 23 &#176;C). The particle size did not change over 6 days (data not shown).
The thermal behavior of the SLNs was investigated using differential scanning calorimetry. The synthesized SLNs have a melting point of 38.0 (&#177; 0.4) and 36.8 (&#177; 0.2) &#176;C for the control SLNs and NiR-loaded SLNs, respectively. In contrast, the melting point for the bulk lipid is 40.0 &#176;C, indicating the suppression of the melting point of the SLNs relative to the bulk lipid. As expected, the confinement to small dimensions and high surface area-to-volume ratio depress the nanoparticle melting point 1. Moreover, the latent heat of melting for the control SLNs and NiR-loaded SLNs is 5.1 (&#177; 0.2), and 4.0 (&#177; 0.1) J/g, respectively. These results indicate that the level of crystallinity of the SLNs is affected by the payload, where the NiR-loaded SLNs showed a lower crystallinity in comparison with the control SLNs.
<img alt="Figure 3" src="/files/ftp_upload/53102/53102fig3highres.jpg" width="600" /> 
Figure 3. Particle size distribution of typical nanoemulsions. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sample</td>
			<td>Particle Size (nm)</td>
			<td>Polydispersity</td>
			<td>Melting Point (&#176;C)</td>
			<td>Latent Heat of Melting (J/g)</td>
		</tr>
		<tr>
			<td>SLNs (Control)</td>
			<td>18.59</td>
			<td>5.83</td>
			<td>38.0 &#177; 0.4</td>
			<td>5.1 &#177; 0.2</td>
		</tr>
		<tr>
			<td>NiR-SLNs</td>
			<td>16.87</td>
			<td>4.47</td>
			<td>36.8 &#177; 0.2</td>
			<td>4.0 &#177; 0.1</td>
		</tr>
	</tbody>
</table>
Table 1. Summary of the physical properties and thermal behavior of the control SLNs and NiR-loaded SLNs including particle size, polydispersity, melting point and latent heat of melting.
In order to explore the interactions of particles and cellular models, the dose limitations of the SLN as directly applied to primary cells in culture, were first established. Using an MTT assay, the dose-response effect on cellular metabolism was measured, where increasing particle concentration resulted in a decrease in cellular viability (with respect to the untreated control cells). There was no observed difference in toxicity between cells exposed to SLN alone versus NiR-loaded SLN at these pre-determined doses. In parallel, cells were visually examined for adherence to the tissue culture polystyrene and images were taken to demonstrate both representative cellular morphology and the uptake of fluorescent particles over time (Figure 5). Using a dose equal to 5 &#956;g/ml lipid (approximately 80% cellular viability) particle uptake was observed by 2 hr post exposure.
<img alt="Figure 4" src="/files/ftp_upload/53102/53102fig4.jpg" /> 
Figure 4. Viability of primary human dermal fibroblasts after 24 hr incubation with nanoparticles. Viability was measured using an MTT assay, and data represent percent viable cells with respect to untreated controls. Concentrations represent weight/volume lipid. Data reflect at least three independent experiments, performed in triplicate. Error bars indicate the standard error of the mean. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53102/53102fig5.jpg" /> 
Figure 5. Primary human dermal fibroblasts after (A) 2 and (B) 24 hr incubation with NiR-loaded nanoparticles. Nanoparticle concentration is equal to 5 &#181;g/ml lipid. Images are representative of at least three independent experiments, performed in duplicate. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Based on the results of the dose limitation studies, the correlation between dose of SLNs and level of incorporation in murine BMDC was investigated. These cells are widely considered the best in vitro model of multiple DC subsets existing in vivo and allow for basic research level investigations of both cellular and molecular effects of intended manipulations. BMDC were left untreated or exposed O/N to either 0.5 or 5.0 &#956;g/ml lipid of the NiR-loaded SLNs (similarly to human fibroblast, use of 50 &#956;g/ml lipid resulted in less than 10% viable cells) and the level of nanoparticle incorporation determined by measuring the intensity of NiR fluorescence in BMDCs via flow cytometry. A direct correlation between the concentration of SLNs used and the amount of fluorescence assessed in BMDCs was observed (Figure 6A). An analysis of the kinetic of NiR-loaded SLNs incorporation revealed a very rapid uptake by BMDC. The SLN fluorescent signal was already evident after 1 hr of exposure, while plateauing at around 5h of exposure (Figure 6B).
<img alt="Figure 6" src="/files/ftp_upload/53102/53102fig6.jpg" /> 
Figure 6. SLN incorporation by dendritic cells correlates with concentration of exposure.&#160;(A) Murine bone marrow-derived dendritic cells (BMDC) were exposed O/N to NiR-SLNs, at the concentration of lipid indicated, and the level of incorporation assessed via flow cytometry. Histogram overlays are all gated on the CD11c+ population. (B) Kinetic of SLN incorporation by BMDC. Cells were exposed to NiR-SLNs (5 &#956;g/ml) for the indicated time and the level of fluorescence (on gated CD11c+ cells) was assessed by flow cytometry. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53102/53102fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications at JoVE.com

Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions.

Solid Lipid Nanoparticles (SLNs) for Intracellular Targeting Applications

Nanoparticle-based delivery vehicles have shown great promise for intracellular targeting applications, providing a mechanism to specifically alter ...

solid-lipid-nanoparticles-slns-for-intracellular-targeting

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

16.8K Views.  The Johns Hopkins Applied Physics Laboratory. In this study, a method for synthesizing ultra-small populations of biocompatible nanoparticles was described, as well as several in vitro methods by which to assess their cellular interactions. 

Video: Solid Lipid Nanoparticles SLNs for Intracellular Targeting Applications

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Cell Labeling and Targeting with Superparamagnetic Iron Oxide Nanoparticles

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the target site while minimizing deleterious effects to off-target sites. Magnetic cell targeting is an efficient, safe, and straightforward delivery technique. Superparamagnetic iron oxide nanoparticles (SPION) are biodegradable, biocompatible, and can be endocytosed into cells to render them responsive to magnetic fields. The synthesis process involves creating magnetite (Fe3O4) nanoparticles followed by high-speed emulsification to form a poly(lactic-co-glycolic acid) (PLGA) coating. The PLGA-magnetite SPIONs are approximately 120 nm in diameter including the approximately 10 nm diameter magnetite core. When placed in culture medium, SPIONs are naturally endocytosed by cells and stored as small clusters within cytoplasmic endosomes. These particles impart sufficient magnetic mass to the cells to allow for targeting within magnetic fields. Numerous cell sorting and targeting applications are enabled by rendering various cell types responsive to magnetic fields. SPIONs have a variety of other biomedical applications as well including use as a medical imaging contrast agent, targeted drug or gene delivery, diagnostic assays, and generation of local hyperthermia for tumor therapy or tissue soldering.

Targeted cell delivery is useful in a variety of biomedical applications. The goal of this protocol is to use superparamagnetic iron oxide nanoparticles (SPION) to label cells and thereby enable magnetic cell targeting approaches for a high degree of control over cell delivery and localization.

Targeted delivery of cells and therapeutic agents would benefit a wide range of biomedical applications by concentrating the therapeutic effect at the ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Manufacture and Drug Delivery Applications of Silk Nanoparticles

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and biodegradability, as well its ability to protect and subsequently release its payload in response to a trigger. While silk can be formulated into various material formats, silk nanoparticles are emerging as promising drug delivery systems. Therefore, this article covers the procedures for reverse engineering silk cocoons to yield a regenerated silk solution that can be used to generate stable silk nanoparticles. These nanoparticles are subsequently characterized, drug loaded and explored as a potential anticancer drug delivery system. Briefly, silk cocoons are reverse engineered first by degumming the cocoons, followed by silk dissolution and clean up, to yield an aqueous silk solution. Next, the regenerated silk solution is subjected to nanoprecipitation to yield silk nanoparticles &#8211; a simple but powerful method that generates uniform nanoparticles. The silk nanoparticles are characterized according to their size, zeta potential, morphology and stability in aqueous media, as well as their ability to entrap a chemotherapeutic payload and kill human breast cancer cells. Overall, the described methodology yields uniform silk nanoparticles that can be readily explored for a myriad of applications, including their use as a potential nanomedicine.

Nanoparticles are emerging as promising drug delivery systems for a broad range of indications. Here, we describe a simple yet powerful method to manufacture silk nanoparticles using reverse engineered Bombyx mori silk. These silk nanoparticles can be readily loaded with a therapeutic payload and subsequently explored for drug delivery applications.

Silk is a promising biopolymer for biomedical and pharmaceutical applications due to its outstanding mechanical properties, biocompatibility and ...

Antimicrobial Characterization of Advanced Materials for Bioengineering Applications

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. Thus, many novel biomaterials are being designed to mimic specific environments required for biomedical applications such as tissue engineering and controlled drug delivery. The development of materials with improved properties for the immobilization of cells or enzymes is also a current research topic in bioprocess engineering. However, one of the most desirable properties of a material in these applications is the antimicrobial capacity to avoid any undesirable infections. For this, we present easy-to-follow protocols for the antimicrobial characterization of materials based on (i) the agar disk diffusion test (diffusion method) and (ii) the ISO 22196:2007 norm to measure the antimicrobial activity on material surfaces (contact method). This protocol must be performed using Gram-positive and Gram-negative bacteria and yeast to cover a broad range of microorganisms. As an example, 4 materials with different chemical natures are tested following this protocol against Staphylococcus aureus, Escherichia coli, and Candida albicans.The results of these tests exhibit non-antimicrobial activity for the first material and increasing antibacterial activity against Gram-positive and Gram-negative bacteria for the other 3 materials. However, none of the 4 materials are able to inhibit the growth of Candida albicans.

We present a protocol for the antimicrobial characterization of advanced materials. Here, the antimicrobial activity on material surfaces is measured by two methods that complement each other: one is based on the agar disk diffusion test, and the other is a standard procedure based on the ISO 22196:2007 norm.

The development of new advanced materials with enhanced properties is becoming more and more important in a wide range of bioengineering applications. ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Applications for Open Source Microplate-Compatible Illumination Panels

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well as large-scale high-throughput screening (HTS) campaigns. Though laboratory automation has greatly increased the utility of microplates there remain instances where automation-based instrumentation is not feasible, cost-effective or compatible with microplate formatting needs. In these cases, microplates must be manually prepared. Problematic to manual microplate manipulations is that a number of difficulties can arise pertaining to the accurate tracking of sample operations, data record keeping and quality control (QC) inspection for well artifacts or formatting errors. As microplate well densities increase (i.e., 96-well, 384-well, 1536-well) the potential for introducing errors also drastically increases.&#160; Moreover, for small bench-top laboratory operations there exists a need to improve the ease and accuracy of sample handling in a cost-effective fashion. Herein, we describe a system that acts as a semi-automated pipetting guide referred to as the Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.). &#160;M.A.P.L.E. has multiple uses for supporting compound hit-picking and microplate preparation for assay development in high-throughput screening or laboratory benchtop operations, as well as QC/quality assurance (QA) diagnostic evaluation of microplate quality or visualizing well formatting errors.

Microplate Assistive Pipetting Light Emitter (M.A.P.L.E.) is a computer-driven device that systematically illuminates microtiter wells to provide guidance for the manual preparation of microplates.&#160; M.A.P.L.E. improves the accuracy of microplate preparation while automating data recordkeeping. &#160;In addition, it can assist with examining microplate quality or aid in the detection of errors.&#160; &#160;

Microplates are commonly used in the modern laboratory environment for a wide variety of tasks both in small-scale laboratory benchtop operations as well ...